Coat Protein-mediated Protection Research Articles

Field Performance of Transgenic Sugarcane Lines Resistant to Sugarcane Mosaic Virus.

Sugarcane mosaic disease is mainly caused by the sugarcane mosaic virus (SCMV), which can significantly reduce stalk yield and sucrose content of sugarcane in the field. Coat protein mediated protection (CPMP) is an effective strategy to improve virus resistance. A 2-year field study was conducted to compare five independent transgenic sugarcane lines carrying the SCMV-CP gene (i.e., B2, B36, B38, B48, and B51) with the wild-type parental clone Badila (WT). Agronomic performance, resistance to SCMV infection, and transgene stability were evaluated and compared with the wild-type parental clone Badila (WT) at four experimental locations in China across two successive seasons, i.e., plant cane (PC) and 1st ratoon cane (1R). All transgenic lines derived from Badila had significantly greater tons of cane per hectare (TCH) and tons of sucrose per hectare (TSH) as well as lower SCMV disease incidence than those from Badila in the PC and 1R crops. The transgenic line B48 was highly resistant to SCMV with less than 3% incidence of infection. The recovery phenotype of transgenic line B36 was infected soon after virus inoculation, but the subsequent leaves showed no symptoms of infection. Most control plants developed symptoms that persisted and spread throughout the plant with more than 50% incidence. B48 recorded an average of 102.72 t/ha, which was 67.2% more than that for Badila. The expression of the transgene was stable over many generations with vegetative propagation. These results show that SCMV-resistant transgenic lines derived from Badila can provide resistant germplasm for sugarcane breeding and can also be used to study virus resistance mechanisms. This is the first report on the development and field performance of transgenic sugarcane plants that are resistant to SCMV infection in China.

Resistance to Alfalfa mosaic virus in transgenic barrel medic lines containing the virus coat protein gene

Medicago truncatula is used as a pasture legume and a source of nitrogen for grain crops in southern Australia. Alfalfa mosaic virus (AMV) infection reduces herbage production and nodulation. The coat protein gene of a South Australian strain of AMV (AMV N20) has been transferred to Medicago truncatula cv. Jemalong 2HA using Agrobacterium-mediated transformation. The most detailed investigations were carried out with the coat protein gene in the sense orientation (CP+). Progeny (T1, T2, T3) raised from self-pollinated primary transformants (T0) containing the coat protein CP+ gene were resistant to AMV. Based on Southern analysis and segregation, the transformants contained a single gene copy. In the T3 generation, one line was immune and one line showed resistance to AMV N20. The immune line contained no detectable virus when plant sap from either inoculated or systemic leaves was bioassayed on Phaseolus vulgaris. This line was also immune to the heterologous AMV S40 isolate. A line with the coat protein gene in antisense orientation (CP–) showed delayed systemic infection but was not immune. We conclude that coat protein mediated protection (CPMP) is an effective strategy for controlling AMV infection and should be further evaluated in the field.

History of coat protein-mediated protection.

A decade of research has proven that plants can be genetically engineered to resist virus infection through expression of viral CP genes, as well as other viral genes and sequences. Additional opportunities for development of resistant plants will require research focused on mechanisms of protection, improvements in expression vector design, and transformation of new crop species. As each of these technologies is utilized singly or in combination to generate resistant crop varieties, the full impact of such engineered resistance will be realized.

The sense and antisense coat protein gene of alfalfa mosaic virus strain N20 confers protection in transgenic tobacco plants

The coat protein gene of a South Australian strain of alfalfa mosaic virus (AMV-N20 [NcS]) has been cloned, sequenced, and transferred into Nicotiana tabacum L. cv. Xanthi via Agrobacterium tumefaciens under the control of the CaMV 35S promoter. A number of lines (T0 generation) were selected with the coat protein gene either in sense orientation (CP+) or in antisense orientation (CP–). The T0 plants were tested for their gene expression and susceptibility to the homologous AMV strain. A significant delay in the onset of symptoms and a reduction in virus accumulation was observed in CP+ plants mechanically inoculated with AMV. CP– plants were also significantly protected but less so than the CP+ plants. Plants transformed with the expression vector only (CP0) showed a minor resistance to local infection on inoculated leaves compared with untransformed plants. The strategy of coat protein mediated protection (CPMP) using the CP gene in either messenger sense or antisense would therefore be appropriate for testing on economically important pasture legumes.

Tobamovirus Cross Protection Using a Potexvirus Vector

Cross protection is the ability of one virus to prevent or delay infection by a related challenge virus. To examine this phenomena, a potato X potexvirus (PVX) vector (Chapmanet al.,1992,Plant J.2, 549) was used to systemically express the tobacco mosaic tobamovirus (TMV) coat protein (CP) open reading frame inNicotiana benthamiana.PVX constructs induce mild mosaic symptoms inN. benthamiana,whereas TMV infection results in rapid systemic necrosis and plant death. Healthy plants or plants preinfected with the unmodified PVX vector succumbed to necrosis within 1 week of TMV challenge inoculation. However, plants preinfected with PVX vectors expressing the TMV CP or RNA coding sequence displayed a 1- to 2-week delay in the appearance of TMV-induced necrosis along with reduced accumulations of TMV. Symptom delay and reductions in the accumulation of TMV were more pronounced in plants protected with the vector expressing the TMV CP than with the vector expressing a nontranslatable CP sequence. Protection was overcome more quickly using a 10-fold higher concentration of challenge inoculum and was ineffective against TMV RNA or a TMV construct expressing a distantly related tobamovirus CP. These findings demonstrate that both RNA and protein are involved in this cross protection phenomenon, but that CP is the main contributor to protection. Taken together, these studies indicate that virus vectors can be used to simulate both cross protection and transgene-derived coat protein-mediated protection, thus providing a new method to further investigate the mechanisms behind each.

Coat protein-mediated protection to cucumber mosaic virus infections in cultivated tomato

Cucumber mosaic virus (CMV) infections rank among the most devastating diseases in the commercial culture of tomato (Lycopersicon esculentum Mill.), for which suitable sources of natural resistance are not available. The concept of pathogen-derived resistance, however, offers an alternate approach to combat plant viral diseases by transformation of crops with nucleotide sequences derived from the viral genome. This report demonstrates the successful application of such a pathogen-derived resistance gene comprising the CMV coat protein (CP) gene, to generate protection to CMV infections in cultivated tomato. Transformation of an inbred tomato line with the CMV CP gene isolated from a subgroup I strain, engendered high levels of protection to various CMV strains, including a virulent strain causing lethal necrosis and a typical subgroup II strain. Moreover, when challenged by natural infection through aphid vectors in open field, levels of protection were largely maintained in hemizygous hybrids. In all, these results demonstrate that synthetic resistance genes based on the CMV CP gene make excellent sources of broad spectrum resistance to CMV infections for introgression into tomato breeding programs.

Biotechnology initiative to achieve plant pest and disease resistance

Biotechnology applications are primarily proprietary, with ∼70% of the annual global research expenditures of US$11 × 10 9 being in the private sector. The International Service for the Acquisition of Agri-Biotech Applications (ISAAA) addresses this challenge by building a new partnership of three donor groups (bilateral/multilateral agencies, private philanthropic foundations, and private companies) to support a not-for-profit institution within existing centres of excellence (Cornell University in North America, John Innes Centre in Europe, Technova in the Asian Pacific Rim, and three additional locations to be established in developing countries). Brokered projects involve only near-term technologies with a high probability of success, using tissue culture/micropropagation, disease diagnostics and transgenic plants. The main targets for transgenic plants are coat protein-mediated protection for non-conventional virus resistance (NCVR) and insecticidal proteins from Bacillus thuringiensis (Bt); both NCVR and Bt can contribute to integrated pest management strategies using reduced inputs for chemical control of insects. Four ISAAA projects demonstrate the potential of NCVR and Bt in developing countries: (1) potato resistant to potato viruses X and Y with Mexico/Monsanto/Rockefeller Foundation, (2) melon resistant to cucumber mosaic virus with Costa Rica/Asgrow Seed/USAID and the ISAAA Fellowship Program, (3) papaya resistant to papaya ringspot virus with Brazil and other countries/Cornell University/Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), and (4) cotton resistant to Lepidoptera with Brazil/EMBRAPA. Although over 1000 field trials of transgenic plants have taken place worldwide, those experiments have been mostly small scale and do not provide sufficient data to assess either the durability of resistance achieved with genetic engineering or conclusive public acceptance of this technology. However, resistance management modelling is speculative and can not decisively indicate a deployment strategy in the absence of continued monitoring and experimentation. ISAAA's projects should allow opportunities for assessment of resistance management practices for applied plant biotechnology to control agricultural pests under diverse production systems.

Coat protein-mediated protection against plum pox virus in herbaceous model plants and transformation of apricot and plum

Coat protein-mediated protection against plum pox virus in herbaceous model plants and transformation of apricot and plum

Coat protein-mediated protection: analysis of transgenic plants for resistance in a variety of crops

Since 1986, research has shown that plants expressing the coat protein gene of a plant virus exhibit degrees of resistance or protection when challenge inoculated with that virus or closely related isolates. This phenomenon, called coat protein-mediated protection, sparked research efforts to develop transgenic plants that resist infection to a range of plant viruses. This report summarizes the research efforts that deal with viral coat protein gene-crop combinations of commercial potential. The viruses include tobacco mosaic, potato virus X and Y, cucumber mosaic and papaya ringspot; the crops include tomato, cucumber, tobacco and papaya.

Cucumber mosaic virus coat protein-mediated protection

Transgenic tobacco plants (CP +) that express the coat protein gene of cucumber mosaic virus (CMV)-Y strain were highly protected from infection with either CMV virions or CMV RNA, while transgenic protoplasts were also protected from infection with CMV virions but not with CMV RNA. CP + plants showed greater susceptibility to infection with satellite RNA-free CMV-Y than CMV-Y containing satellite RNA. At temperatures above 30°C, CP + plants did not or poorly resist infection with CMV. Elevated temperature affected the accumulation of CP rather than its mRNA, suggesting that CP molecules are mainly involved in virus resistance in CP + plants.

CDNA sequence of the pepper severe mosaic virus coat protein gene.

PeSMV was originally isolated in Argentina from diseased Capsicum annuum plants and classed as a possible member of the potyvirus group on the basis of morphological, biological, and immunological evidence (Feldman and Gracia, 1977). Although information concerning its economic significance is still imprecise, the virus is present in most pepperproducing areas and yields are drastically reduced in chronically infected plants. As part of a project to obtain resistant transgenic plants by means of coat protein-mediated protection, we cloned the PeSMV gRNA and obtained a partia1 nucleotide sequence of the PeSMV genome including the 3’ end of the replicase gene, the complete CP gene, and the 3’ noncoding region (Table I). PeSMV gRNA from strain 5-163 was used for cDNA synthesis. Clones located in the genome 3‘ end were identified by low-stringency hybridization with a radioactive PVY CP probe and dideoxy sequenced. A sequence spanning 1329 nucleotides from the 3‘ end was obtained, the first 998 encoding a continuous open reading frame. Comparison of the sequence encoding the putative PeSMV CP with that of PVY revealed a similarity of 71.6%. The 3’ noncoding sequences are different lengths (PeSMV, 471 nucleotides; PVY, 331 nucleotides) and were not directly comparable. The translated amino acid sequence corresponding to the putative PeSMV CP showed similarities of 80.9 and 62.1% with the PVY (Bravo-Almonacid and Mentaberry, 1989) and tobacco etch virus (Allison et al., 1985) CPs, respectively. Most of the sequence changes are clustered at the N-terminal region. The PeSMV sequence contains two additional codons between nucleotides 142 and 147 of the PVY CP sequence. On the other hand, severa1 sequence motifs strongly conserved in most potyvirus are present in the C-terminal threequarters of the sequence. Remarkably, the PeSMV sequence does not contain the amino acid motif DAG that was associated with aphid transmissibility (Atreya et al., 1991). This fact agrees with previous data showing that PeSMV is not aphid transmitted (Feldman and Gracia, 1977).

Strategies to protect crop plants against viruses: pathogen-derived resistance blossoms.

Since 1986, the ability to confer resistance against an otherwise devastating virus by introducing a single pathogen-derived or virus-targeted sequence into the DNA of a potential host plant has had a marked influence on much of the research effort, focus, and short-term objectives of plant virologists throughout the world. The vast literature on coat protein-mediated protection, for example, attests to our fascination for unraveling fundamental molecular mechanism(s), our (vain) search for a unifying hypothesis, our pragmatic interest in commercially exploitable opportunities for crop protection, and our ingenuity in manipulating transgene constructions to broaden their utility and reduce real or perceived environmental risk issues. Other single dominant, pathogen-derived plant resistance genes have recently been discovered from a wide variety of viruses and are operative in an ever-increasing range of plant species. Additional candidates seem limited only by the effort invested in experimentation and by our ingenuity and imagination. This review attempts to consider, in a critical way, the current state of the art, some exceptions, and some proposed rules. The final impression, from all the case evidence considered, is that normal virus replication requires a subtle blend of host- and virus-coded proteins, present in critical relative concentrations and at specific times and places. Any unregulated superimposition of interfering protein or nucleic acid species can, therefore, result in an apparently virus-resistant plant phenotype.

Virus Resistant Papaya Plants Derived from Tissues Bombarded with the Coat Protein Gene of Papaya Ringspot Virus

Papaya ringspot virus (PRV) is a serious disease of papaya (Carica papaya L.) that has only been partially controlled by conventional methods. An alternative control method is coat protein-mediated protection (CPMP) through the transfer and expression of the PRV coat protein (cp) gene in papaya. We report an efficient gene transfer system utilizing microprojectile-mediated transformation of 2,4-D-treated immature zygotic embryos with a plasmid construction that contains the neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS) genes flanking a PRV cp gene expression cassette. Putative transgenic RO papaya plants, regenerated on kanamycin-containing medium, were assayed for GUS and PRV coat protein expression, for the presence of NPTII and PRV cp genes [with the polymerase chain reaction (PCR) and genomic blot hybridization analysis], and for PRV cp gene transcripts by Northern analysis. Four RO transgenic plant lines that contained the PRV cp gene showed varying degrees of resistance to PRV, and one line appeared to be completely resistant. These results represent the first demonstration that CPMP can be extended to a tree species such as papaya.

Expression of cowpea mosaic virus coat protein precursor in transgenic tobacco plants

Tobacco, Nicotiana tabacum L., supports cowpea mosaic virus (CPMV) replication and cell-to-cell movement, and thus may serve as a model system to study coat protein-mediated protection against CPMV. A chimeric gene consisting of the cauliflower mosaic virus 35S promoter, CPMV 60K coat proteins-precursor (CP-P) coding region, and the nopaline synthase polyadenylation signal was transferred to tobacco cv. Burley 21 via the Agrobacterium tumefaciens binary vector system. Gene integration and expression in the transgenic tobacco plants were confirmed by Southern and RNA dot blot analyses. Accumulation of CPMV 60K CP-P in transgenic plants, up to 2 micrograms/g of wet weight tissue, was detected by ELISA and Western blots. The results of Western blots and immunosorbent electron microscopy further indicated that CPMV CP-P neither undergoes autoproteolysis to generate the mature viral coat proteins nor assembles into virus-like capsids, suggesting that processing of the CP-P may be required for virus assembly. Because CPMV neither induces symptoms in tobacco nor moves systemically, evaluation of the reactions of the transgenic plants to virus inoculation was based on virus accumulation in the inoculated leaves. Results from such infectivity experiments did not differentiate between CP-P expressers and vector-transformed plants. The transgenic tobacco plants expressing CP-P should provide valuable material for investigating comovirus polyprotein processing and capsid assembly in vivo.

The second amino acid of alfalfa mosaic virus coat protein is critical for coat protein-mediated protection.

Transgenic plants expressing the coat protein (CP) of alfalfa mosaic virus (AIMV) are resistant to infection by AIMV. A mutation was introduced into the second amino acid of the cDNA for the CP of AIMV. Three different transgenic tobacco lines expressing the mutant CP and two different transgenic tobacco lines expressing the wild-type CP at similar levels were challenged with AIMV virions and viral RNA. Whereas the lines expressing the wild-type CP were highly resistant to infection by AIMV virions and viral RNA, the lines expressing the mutant CP were susceptible to infection by both. The binding affinity of the mutant and the wild-type CPs for the 3' terminal protein binding site on AIMV RNAs was similar, as determined by electrophoretic mobility shift assay. A mixture of AIMV genomic RNAs 1-3 was infectious on the plants expressing the mutant CP but not on vector control plants or plants expressing the wild-type CP, indicating that the mutant CP can activate the AIMV genomic RNAs for infection. These results demonstrate that the second amino acid of the AIMV CP is critical for protection from AIMV but not for the initial interaction between the AIMV RNA and CP, suggesting that this initial interaction does not play a major role in CP-mediated protection.

Susceptibility to virus infection of transgenic tobacco plants expressing structural and nonstructural genes of tobacco rattle virus

Tobacco plants were transformed with the coat protein (CP) genes and several nonstructural genes of tobacco rattle virus (TRV) strains PLB and TCM. Accumulation of RNA transcripts from the integrated viral genes was detectable in all types of transformants. Plants expressing CP were resistant to infection with virions of the homologous strain but susceptible to infection with RNA of the homologous strain or nucleoprotein of the heterologous strain. No resistance was detectable in plants transformed with the nonstructural 13K and 16K genes of strain PLB, or with the 29K gene that is unique to RNA-2 of strain TCM. When protoplasts from plants expressing TCM-CP were inoculated with TCM virions, there was a normal production of genomic RNAs and CP but the synthesis of mRNA and protein corresponding to the 16K gene was selectively defective. Because this defect was not observed when protoplasts from plants expressing PLB-CP were inoculated with PLB virions, it probably plays no role in the coat protein-mediated protection observed in transgenic plants.

Resistance in Transgenic Potato Expressing the Potato Leafroll Virus Coat Protein Gene

In an attempt to obtain coat protein-mediated protection, the coat protein gene of PLRV, which was previously identified and sequenced, was inserted into the genomes of both tobacco and potato. These plants were characterized, the potato plants challenged with PLRV using viruliferous aphids, and virus titers monitored using ELISA. The results were compared to those of the transgenic plants expressing the coat protein of other viruses

Effect of protein aggregation state on coat protein-mediated protection against tobacco mosaic virus using a transient protoplast assay

To address the mechanism(s) of protection against tobacco mosaic virus (TMV) infection conferred by expression of the TMV capsid protein (CP) gene in transgenic tobacco plants, a transient protection assay has been developed. Introduction of either purified viral CP or virus inactivated by ultraviolet irradiation into tobacco protoplasts induced a transient protection to challenge virus introduced concomitantly or shortly thereafter. The transient protection was characterized and the effects of different aggregation states of TMV CP were tested in the transient assay system. Tobacco mosaic virus CP preparations composed largely of helical, virus-like, aggregates conferred a less transient protection against TMV and greater protection against a distantly related virus than did preparations composed primarily of smaller aggregates.

Engineering virus resistance in agricultural crops

Plant viral genomes are relatively small and in the past decade many have been characterized at the molecular level. This has prompted research into the development of virus resistance based on interference with the viral multiplication cycle by teh introduction of viral sequences into the plant genome. Several strategies have been tested. The most successful one so far involves the constitutive expression of the coat protein gene of the virus against which resistance is desired. In this review we describe progress made in engineering virus resistance into potato, an important agricultural crop. To this end the molecular structure of the potato viruses X and Y and leafroll is discussed as well as the introduction of resistance against potato virus X into potato. In addition, we address the question of preservation of cultivar-specific characteristics, an important prerequisite for commercial application. Finally, recent investigations for alternative forms of virus resistance are described against the background of the results of coat protein-mediated protection.